This invention relates generally to the targeted activation of biologically active materials to cells that produce prostate specific antigen (PSA) and more specifically to PSA-specific peptides that activate therapeutic drugs. The invention also describes analogs of a unique cytotoxic agent, thapsigargin. In these analogs the TG molecule has been modified such that a primary amine containing side chain has been introduced to allow for coupling to the C-terminal carboxyl of a peptide.
There is currently no effective therapy for men with metastatic prostate cancer who relapse after androgen ablation, even though numerous agents have been tested over the past thirty years. Prolonged administration of effective concentrations of standard chemotherapeutic agents is usually not possible because of dose-limiting systemic toxicities.
Prostate specific antigen (PSA) is a 33,000 kDa single chain glycoprotein first characterized from human prostate tissue. PSA is synthesized and secreted as a unique differentiation product of the prostatic glandular cells, both from normal and cancerous cells. Low levels of PSA are detected in normal and cancerous breast tissue also.
PSA is a serine protease with extensive sequence identity to the glandular kallikreins. It has chymotrypsin-like substrate specificity. Major proteolytic substrates are gel-forming proteins in freshly ejaculated semen, semenogelin I (SgI) and semenogelin II (SgII), produced in the seminal vesicles. Other PSA substrates are extracellular matrix components fibronectin, laminin, insulin-like growth factor binding proteins, the single chain form of urokinase-type plasminogen activator, and parathyroid hormone-related protein. PSA is enzymatically active in the extracellular fluid of prostatic cancer while enzymatically inactivated in the blood serum.
Thapsigargin (TG) is a sesquiterpene-xcex3-lactone available by extraction from the seeds and roots of the umbelliferous plant Thapsia garganica L. Thapsigargin selectively inhibits the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) pump, found in skeletal, cardiac, muscle and brain microsomes. The apparent dissociation constant is 2.2 pM or less.
TG operates by what is believed to be a unique method of killing cells. TG induced inhibition of the SERCA pump leads to depletion of the ER Ca2+ pool. This depletion apparently results in the generation of a signal, possibly from an ER-derived diffusible messenger, so that the plasma membrane is more permeable to extracellular divalent cations. The resulting influx of these cations is responsible for the death of cells.
TG is poorly soluble in water, does not possess cell specificity, and is able to kill quiescent Go cells. For these reasons, unmodified TG would be difficult to administer and deliver systemically without significant non-specific host toxicity.
The present invention provides a novel class of peptides that include amino acid sequences containing cleavage sites for prostate specific antigen (PSA) and other enzymes with the same activity and proteolytic specificity as PSA. A representative amino acid sequence is provided and includes Ser-Lys-Leu-Gln (SEQ ID NO:1), analogs, derivatives and conservative variations thereof.
The invention also provides novel analogs of therapeutic sesquiterpene-xcex3-lactones, including derivatives of the thapsigargins. The thapsigargins are a group of natural products isolated from species of the umbelliferous genus Thapsia. The term thapsigargin has been defined by Christensen, et al., Prog. Chem. Nat. Prod., 71 (1997) 130-165. These derivatives contain a means of linking the therapeutic drug to carrier moieties, including peptides and antibodies. The peptides and antibodies can include those which specifically interact with antigens including PSA. The interactions can involve cleavage of the peptide to release the therapeutic analogs of sesquiterpene-xcex3-lactones.
The invention also provides a therapeutic prodrug composition, comprising a therapeutic drug linked to a peptide which is specifically cleaved by PSA. The linkage substantially inhibits the non-specific toxicity of the drug, and cleavage of the peptide releases the drug, activating it or restoring its non-specific toxicity.
The invention also provides a method for treating cell proliferative disorders, including those which involve the production of PSA, in subjects having or at risk of having such disorders. The method involves administering to the subject a therapeutically effective amount of the composition of the invention.
The invention also provides a method of producing the prodrug composition of the invention. In another embodiment, the invention provides a method of detecting PSA activity in tissue. In yet another embodiment, the invention provides a method of selecting appropriate prodrugs for use in treating cell proliferative disorders involving PSA-production.
The invention also provides a method for detecting a cell proliferative disorder associated with PSA production in a tissue of a subject, comprising contacting a target cellular component suspected of having a PSA associated disorder, with a reagent which detects enzymatically active PSA.
The invention also provides a method of determining PSA activity in a PSA-containing sample, comprising contacting the sample with a detectably labeled peptide which is specifically cleaved by PSA for a period of time sufficient to allow PSA to cleave the peptide, detecting the detectable label to yield a detection level, which is then compared to the detection level obtained by contacting the same detectably labeled peptide with a standard PSA sample of known activity.
The invention also provides a method of imaging soft tissue and/or bone metastases which produce PSA, comprising administering a lipophilic imaging label linked to a peptide which is specifically cleaved by PSA to a subject having or suspected of having a PSA-associated cell proliferative disorder, allowing PSA to cleave the peptide, allowing the lipophilic imaging label to accumulate in the tissue and/or bone, allowing the subject to clear the uncleaved peptide, and imaging the subject for diagnostic purposes.
Unless otherwise defined, all technical and scientific terms used herein have the ordinary meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other reference materials mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.